Measuring Bacterial Growth by Quantitative Chloroplast Estimation

Introduction

This set of instructions explains how to measure the number of chloroplasts in a given amount of solution. The measurements can be extrapolated to determine the amount of growth in bacteria. Bacterial cultures grow at different rates depending on their concentration. At first, when there are few bacteria in the population, the culture grows slowly. But in what is called the log phase, the culture grows very quickly. Then, as the bacteria overcrowd and as resources become scarce, the growth of the bacterial culture slows down. Research on bacterial growth in the log phase is the most useful. If the literature does not already hold information on bacterial growth in the log phase, the scientist conducting the experiment must experimentally figure out when the log phase occurs. The procedure outlined in this document will enable a scientist to identify the log phase timing of a bacterial culture grown in a liquid medium.

Notes and Cautions

This is important scientific data and research. Be sure to refer to the instruction and procedures manuals of all equipment. Always wear gloves and eye protection. This process usually takes 30-45 minutes for three samples. In order to obtain an accurate graph of the growth phases of your bacteria, this process must be repeated every few (2-3) days.

• Cyanobacteria (a bacteria with chloroplasts)
• A medium to grow the bacteria in (solution of nutrients designed to feed a growing bacteria population)
• An incubator (controlled environment that enables bacteria to grow)
• 1mL (1000µL) pipette
• 1mL (1000µL) pipette tips
• Microcentrifuge (a machine that spins microcenterfuge tubes at high speeds to separate particles by centrifugation)
• Microcentrifuge tubes
• Vortex (a plate which spins in small circles at high speeds, creating a vortex within liquid samples)
• Methanol
• Cuvettes
• Spectrophotometer (a machine that measures the absorbance of light at specific wavelengths)
• Biomaterial disposal container

Figure 1: Preventing Cross-Contamination

The bacteria used in this example is a culture of Anabaena variabilis growing in 50 mL of the recommended media (BG-11o). Different species of bacteria grow differently in different media and under other conditions, but finding the correct growth conditions for any given bacteria is beyond the scope of this instructable. In our experience with this particular bacteria, 50 mL is enough media that taking a 1 mL sample will not dramatically affect the bacteria’s ability to continue growing nor overconcentrate the bacteria within the media, which would also slow the growth rate of the bacteria.

Take your 1mL pipette, attach a 1 mL pipette tip, and draw up 1 mL of your sample. Dispense the sample into a microcentrifuge tube. Discard the pipette tip and close the microcentrifuge tube. If you are measuring more than one sample, always discard tips and close tubes between each sample. It is imperitive for accurate readings that you do not cross-contaminate. See figure 1.

Figure 2: Centrifuges: Separating Particles by Density

Put the microcentrifuge tube into a microcentrifuge. Add a centrifuge tube with 1mL of water to balance the microcentrifuge, if necessary. Close the microcentrifuge and set it to run at full speed for 5 minutes. See figure 2. When the microcentrifuge has finished spinning, take out as much of the media around the bacteria as possible with the 1mL pipette (with a new tip for each sample). Discard the media in an appropriate biomaterial waste container.

Figure 3: Releasing Chloroplasts from their Dead Host Bacteria

Add 1mL of methanol to the microcentrifuge tubes. Make sure to cap the methanol container when it is not in use. Methanol is a volatile chemical, which means that it will evaporate at room temperature. Vortex all the samples to make sure all the cells are broken and the chloroplasts are released. This step is especially important if there is anything in the media with the bacteria (such as dirt or catalytic metals). Follow the instructions on your vortex to use it, though most are activated, when turned on, by pressing your tube down onto the plate. See figure 3.

We strongly recommend using a vortex to break the cells open. However, if a vortex is not available, this step can also be done by vigorously shaking the microcentrifuge tubes. While vortexing may take only seconds, we recommend shaking for at least 5 minutes. Any visible particles should come off the sides and bottom of the tube. If you are also using cyanobacteria, the sample should lose its green color and become blue.

Figure 4: Collecting Spectral Data

With the chloroplasts properly released, the solution at this point can be placed in a small, square vial called a cuvette. This cuvette is then placed in a spectrophotometer. A properly calibrated spectrophotometer will return a measurement of optical density, or absorbance, at the target wavelength. This number can be used on its own as a reference. Otherwise, it can be used to measure the amount of chlorophyll or to estimate the number of bacterial cells in the sample. The optical density multiplied by 13.43 will produce the micrograms of chlorophyll per mililiter in the sample. The optical density multiplied by 4.3∗10^7 (43,000,000) will offer a rough estimate of cells per mililiter in the sample. See figure 4.

Successive samples can (and should) be taken in this manner as the bacterial culture continues to grow. Plotting the data taken over the course of a few weeks will reveal the log phase of the bacterial culture.